Thrombin, a key enzyme in hemostasis, has both procoagulant and anticoagulant properties, based on its different substrate specificities. Thrombin is secreted from the liver as an inactive zymogen, prothrombin, that is activated by coagulation factors Va and Xa to yield mature α-thrombin. This process can be mimicked in vitro by the proteolytic cleavage of prothrombin with various snake venoms such as Echis carinatus venom.
Thrombin acts as a procoagulant by the proteolytic cleavage of fibrinogen, ultimately resulting in the formation of an insoluble fibrin clot, the activation of the clotting cofactors factor V and Factor VIII to FVa and FVIIIa, the cleavage of Factor XI to activated Factor XIa (leading to further activation of Factors IX and X and perpetuation of clotting) and the cleavage of the platelet thrombin receptor, resulting in platelet activation. On the other hand, when thrombin binds to thrombomodulin (TM), an integral membrane protein on vascular endothelial cells, thrombin undergoes a conformational change such that thrombin loses its procoagulant activity and instead acquires the ability to convert a plasma protein called protein C (PC) to activated protein C (aPC). aPC, a serine protease, acts as a potent anticoagulant by inactivating activated FV (FVa) and FVIII (FVIIIa), two essential cofactors in the clotting cascade. aPC also inactivates plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of tPA (tissue plasminogen activator), thus potentiating normal fibrinolysis. This mechanism may serve to ensure that blood coagulation remains localized at the site of injury. Infants completely deficient in PC are essentially incompatible with life, with a fatal thrombotic disorder called neonatal purpura fulminans; some patients with a partial deficiency of PC have recurrent thrombosis. In addition, many recent animal models utilizing aPC infusion have shown that exogenous aPC is an anti-thrombotic and anti-inflammatory molecule.
Human thrombin is generated from a precursor polypeptide, prothrombin, of approximately 579 mature amino acids (subject to potential allelic variation or N-terminal microheterogeneity) plus a presequence of about 43 residues (Degen et al., “Biochemistry” 22:2087 [1993]). The presequence is proteolytically removed by the cell during the process of expression and secretion of prothrombin. Prothrombin is a zymogen, or inactive protease, that is activated by proteolytic cleavage. At least three basic sites are subject to cleavage. In vivo, prothrombin is cleaved between residues R271 and T272 (Degen et al. residue numbers) by Factor Xa in the presence of Factor Va, phospholipid and calcium ions to yield prethrombin 2 and Fragment 1.2. Prothrombin also is proteolytically cleaved by the same system between R320 and I321 to yield meizothrombin, which in turn is cleaved autolytically between R155 and S156 to produce Fragment 1 (1–155) and meizothrombin des 1 (a disulfide linked dipeptide extending from residue 156 to the carboxy terminus of prothrombin, cleaved at R323). Finally, thrombin is generated from prethrombin 2 by proteolytic cleavage between R320 and I321, or from meizothrombin des 1 by proteolytic cleavage between R271 and T272. Thrombin itself then autolyzes cleavage between T284 and T285 to generate the mature A-chain N-terminus. For the purposes herein, the mature N-terminal residue of the thrombin A chain (Degen T285) is designated “T1a” and is then numbered consecutively to the arginine residue at R36a. The B chain is numbered from its N-terminal residue I1 (Degen I321) through E259. The two thrombin peptides are covalently bonded by a disulfide linkage between C9a and C119.
Two distinct numbering systems are in use for thrombin, in addition to the DNA-based system of Degen et al. One is based on alignment with chymotrypsinogen (Bode et al., “EMBO J” 8:3467 (1989). A second is favored by Sadler and coworkers at the University of Washington. The Sadler numbering scheme is used in this specification. Under this protocol, the B chain of thrombin commences with I1 and extends to E259, while the A chain is designated with “a” postscripts as noted above, as in T1a to R36a. This thrombin is termed “reference sequence thrombin,” and its entire sequence is shown in FIG. 1. For example, Wu et al., (“PNAS USA” 88:6775, (1991)) disclose several thrombin mutants numbered in accordance with the Sadler scheme. The Wu et al. mutants and the corresponding chymotrypsinogen and Degen et al. residue numbers, respectively, are sequentially shown as follows: H43 (57, 363), K52 (60f, 372), N53 (60 g, 373), R62 (67, 382), R68 (73, 388), R70 (75, 390), D99 (102, 419) and S205 (195, 525). SEQ. ID. NO. 2 uses the Sadler system also, but the first residue is the A-chain residue. Numerically, therefore, the residue numbers of SEQ. ID. NO. 2 (and the claims) are shifted 36 higher than elsewhere in this specification.
It is known in the literature that the thrombin binding sites for fibrinogen and protein C activation are overlapping but not identical. This is based on a small number of thrombin mutants (Wu et al., op cit). Wu et al. reported that a polypeptide having the sequence of thrombin but with glutamic acid substituted at position 52 (K52E) was approximately 2.5 fold more active in producing activated PC than wild-type thrombin and possessed only about 17% of the normal fibrinogen clotting activity of wild-type thrombin. Conversely, a polypeptide having the sequence of thrombin but with glutamic acid substituted at position 70 (R70E) reportedly had the fibrinogen clotting activity of wild-type thrombin but only approximately 7% of the PC activating capability of wild-type thrombin. According to Wu et al., the R68E protein essentially lost both functions.
For other polypeptides having sequence homology to thrombin, see Le Bonniec et al., “JBC” 268(25):19055 (1993); Le Bonniec et al., “JBC” 266(21):13796 (1991); Le Bonniec et al., “PNAS USA” 88:7371 (1991); Sheehan et al., “JBC” 268(5):3639 (1993); Horrevoet et al., “JBC” 268(2):779 (1993); Suzuki et al., “JBC” 266(28):18498 (1991); Sheehan et al., “Thrombosis and Haemostasis” 69:Abstract 1784 (1993); Gan et al., “Thrombosis and Haemostasis” 69:Abstract 1783 (1993); Gan et al., “Thrombosis and Haemostasis” 69:Abstract 1787 (1993), and Naray-Szabo et al., “Theochem” 59:401 (1989).
The pivotal role of thrombin in blood clotting has made this protein a target in the development of agents for the treatment of thrombosis. Most efforts have focused on the direct inhibition of the thrombin proteolytic activity, and in fact numerous inhibitors of the procoagulant activities of thrombin have an anticoagulant effect (Hirsh, 1991; Hirsh, 1991a). However, the potency of these inhibitors may be limited by the concomitant inhibition of the anticoagulant activity of thrombin. Conversely, anticoagulant effect has been achieved by augmenting or stimulating the thrombin anticoagulant pathway, i.e., by administration of soluble TM (Gomi, et al., “Blood” 75:1396–1399, 1990; and Light, D., WO 93/15755) or activated protein C (“aPC”) Dreyfus et al., 1991; Gruber et al., 1990; Gruber et al., 1991; Taylor et al., 1987). This strategy does not block ongoing coagulation resulting from previously activated thrombin.
It is an object of this invention to prepare novel polypeptides which possess enhanced physiochemical or biological activities.
A further object of this invention is to prepare novel polypeptides in which the procoagulant and anticoagulant activities of thrombin have been substantially segregated, and which also optionally resist heparin-mediated antithrombin III (AT-III) inhibition.
Another object is to obtain such polypeptides that can be expressed in elevated yields in recombinant cell culture.
An additional object of this invention is to provide novel polypeptides that are substrate specific for protein C activation or fibrinogen, but do not substantially proteolyze polypeptides that normally are not thrombin substrates.
Another object of this invention is to provide novel covalently-modified polypeptides that are useful in screening for substances that are agonists or antagonists of thrombin's procoagulant or anticoagulant activities.
A further object is to provide novel polypeptides that activate protein C but are substantially incapable of, or have reduced proteolytic activity against any one or more of fibrinogen, the thrombin platelet receptor, and/or Factors XIII, V, XI or VIII.
A still further object is to obtain novel polypeptides useful in purifying thrombin-interactive polypeptides such as TM or aPC from cell culture or native sources.
Another object is to identify novel polypeptides retaining at least a substantial degree of the desired proteolytic activity of thrombin, including the kcat and Km of thrombin for the desired substrate.
In a further object, novel polypeptides are provided that exhibit enhanced PAI-1 inactivating activity as compared to wild-type thrombin.
A further object is to provide a method for identifying deficiencies in thrombomodulin function in patients with clotting disorders.
In other objects, novel polypeptides are provided for the treatment of thrombosis, in particular thrombosis associated with septic shock, for the therapy of solid tumors and for the preparation of improved dressings for wounds, or for therapies and diagnostic utilities that rely upon a property of thrombin.
Another object is to identify novel analogues of thrombin that have an enhanced or reduced ability to stimulate cell proliferation (Ben-Sharit et al., “PNAS USA” 83:976–980 (1986)).
These and other objects of the invention will be apparent from consideration of this specification as a whole.